The prostate is almost invariably the site of benign and malignant proliferative changes in aging males. Prostatic intraepithelial neoplasia (PIN) develops into prostate cancer in a significant percentage of men, thereby indicating that it might be a premalignant lesion (Bostwick D G and Brawer M K, Cancer 59:788-794, 1987). The relationship between PIN and prostate cancer has been reported in multiple studies (Sakr W A, Grignon D J, Crissman J D, Heilbrun L K, Cassin B J, Pontes J J, Haas G P, In Vivo 8:439-443, 1994; Bostwick D G, Qian J, Am J Surg Pathol 19:506-518, 1995(b); Arakawa A, Song S, Scardino P T, Wheeler T M, Pathol. Res. Pract. 191:868-872, 1995; Qian J, Bostwick D G, Pathol. Res. Pract. 191:860-867, 1995; Qian J, Wollan P, Bostwick D G, Hum. Pathol. 28:143-148, 1997). In fact, the multifocality of PIN within the prostate parallels that of adenocarcinoma and can be traced to the same zones within the gland, for example, the peripheral zone, thus providing a spatial linkage between the two entities (Bostwick, D G, Cancer 75:1823-1836, 1995(a); Kovi J, Mostofi F K, Heshmat M Y, Enterline J P, Cancer 61:555-561, 1988; Bostwick D G, Amin M B, Dundore P, Marsh W, Schultz D S, Hum Pathol. 24:298-310, 1993; Bostwick, D G, Cancer 75:1823-1836, 1995(a); Qian J, Wollan P, Bostwick D G, Hum. Pathol. 28:143-148, 1997). Further, only about one-third of radical prostatectomy specimens have high-grade PIN in both the peripheral and transition zones and when carcinoma is found in the transition zone, there is usually concurrent PIN present.
Data from autopsy studies also supports a relationship between PIN and prostate cancer. For example, in a series of 249 autopsy cases, 77% of prostates with PIN harbored invasive adenocarcinoma, compared to only 24% without PIN (Sakr W A, Grignon D J, Crissman J D, Heilbrun L K, Cassin B J, Pontes J J, Haas G P, In Vivo 8:439-443, 1994). Autopsy studies also demonstrate that development of PIN predates the development of clinically detectable cancer by 5 to 10 years, consistent with the concept that PIN is a pre-malignant lesion (Orozco R, O'Dowd G, Kunnel B, Miller M C, Veltri R W, Urology 51:186-195, 1998). Silvestri et al performed an autopsy study of European men and found an association between PIN and carcinoma in the majority of cases (Silvestri F, Bussani R, Pavletic N, Bassan F, Pathol. Res. Pract. 191:908-916, 1995). However, more recent analyses have revealed that 70% of autopsy cases with histologic evidence of invasive prostate do not have associated PIN. In a series of 62,537 needle biopsies of the prostate, isolated PIN was found in only 4.1% of cases while invasive cancer was found in 38.3% of cases (Orozco R, O'Dowd G, Kunnel B, Miller M C, Veltri R W, Urology 51:186-195, 1998). Collectively, these findings suggest that a subset of prostate cancers may develop de novo and do not begin with PIN.
Franks (Franks, L. M. (1954) J. Pathol. Bacteriol. 98:617-621) first proposed that atypical hyperplasia was a precursor of prostatic carcinoma. Helpap (Helpap, B. (1980) Virch. Arch. 387:307-31) demonstrated by 3H-thymidine uptake that “severe atypical primary hyperplasia is a precancerous lesion”. In a key study, McNeal and Bostwick (McNeal et al. (1986) Hum. Pathol. 17:64-71) showed an association between high grade prostatic intraepithelial neoplasia (HGPIN) and prostatic adenocarcinoma. Bostwick (Bostwick, D. G. et al. (1987) Cancer 59:788-794) subsequently provided a detailed description of the architectural features of HGPIN where he pointed out that >75% of all HGPIN exhibits a dome-like architecture. Since these initial studies, Epstein (Epstein, J. I. et al. (1990) Cancer 65:2321-2327) provided further linkage of HGPIN with organ-confined carcinoma, and multiple studies have now described HGPIN and demonstrated a significant association with cancer. In a series of 249 autopsy cases, 77% of prostates with HGPIN harbored invasive adenocarcinoma, compared to only 24% without HGPIN. Autopsy studies also demonstrated that development of HGPIN predated the development of clinically detectable cancer by 5 to 10 years, consistent with the concept that HGPIN is a pre-malignant lesion. Further, studies have also demonstrated correlation of low-grade prostatic intraepithelial neoplasia (LGPIN) with the development of malignant lesions (Goeman et al. (2003) Prostate Cancer Prostatic Dis. 6:305-10).
Other studies have provided strong support for the association of HGPIN with of the incidence of PCA. For example, a group retrospectively identified 190 men with HGPIN, and 1677 men with only benign prostatic in needle biopsy tissue. The cumulative risk of detection of carcinoma on serial sextant follow-up biopsies was 30.5% for those with isolated HGPIN compared with 26.2% for the control group. HGPIN found on the first repeat biopsy was associated with a 41% risk of subsequent detection of carcinoma compared with an 18% risk if benign prostatic tissue was found on the first repeat biopsy. The results suggest that the risk of prostate carcinoma is 30.5% after a diagnosis of isolated HGPIN in a needle biopsy. Likewise, another group showed that the multiple core involvement by high grade PIN or HGPIN, both on initial and first repeat biopsy, defines a subset of men that are at increased risk of harboring synchronous invasive carcinoma. Other studies have demonstrated that high grade PIN, patient age and PSA are all highly significant predictors of PCA with PIN having the highestrisk ratio. In fact, PIN has been shown to be more predictive of PCA in older patients and those with a serum PSA of >4 ng/ml. Since PIN predates the appearance of PCA by ˜2-5 years these reports suggest that patients with high grade PIN need to aggressively monitored for the development of cancer.
A common method of diagnosis for prostate cancer is determining the level of prostate specific antigen (PSA) in the blood. PSA is a glycoprotein secreted by the prostate gland. The PSA test does have limitations of sensitivity and selectivity: in general, levels above 4 ng/ml are suggestive of cancer and levels above 10 ng/ml are highly suggestive. Also, many persons with prostate cancer have normal PSA levels at the time of diagnosis. Therefore, molecular markers with greater sensitivity and selectivity for prostate cancer and the premalignant condition of PIN would be useful for, among other things, the diagnosis of these conditions.
The ATP-binding cassette (ABC) transporter superfamily is one of the largest gene families comprising at least 48 genes and encoding a functionally diverse group of membrane proteins involved in energy-dependent transport of a wide variety of substrates across membranes (Dean et al., Curr Opin Genet Dev, 1995, 5, 779-85). It constitutes a family of proteins that are extremely well conserved during evolution, from bacteria to humans (Ames and Lecar, FASEB J., 1992, 6, 2660-2666). Physiological studies have shown that the prototype ABC protein binds ATP and uses the energy from ATP hydrolysis to drive the transport of various molecules across cell membranes. Most ABC functional proteins from eukaryotes encode a full-transporter, each consisting of two ATP-binding domains (nucleotide binding fold, NBF) and two transmembrane (TM) domains, most of which are arranged in a TM-NBF-TM-NBF fashion (Dean et al., Curr Opin Genet, 1995, 5, 79-785). So far, it is known that the ABC-binding cassette proteins are involved in extracellular and intracellular membrane transport of various substrates, including ions, amino acids, peptides, sugars, vitamins, cholesterol or steroid hormones.
Among the 48 identified human ABC transporter members, 11 have been associated with human disease, including ABCA1, ABCA4 (ABCR) and ABCC7 (CFTR), which are thought to be involved in Tangier disease (Bodzioch M et al., Nat. Genet., 1999, 22(4); 347-351; Brooks-Wilson et al., Nat. Genet., 1999, 22(4), 336-345; Rust S et al., Nat. Genet., 1999, 22, 352-355; Remaley A T et al.,), Stargardt disease (Lewis R A et al., Am. J. Hum. Genet., 1999, 64, 422-434), and cystic fibrosis (Riordan J M et al., Science, 1989, 245, 1066-1073), respectively. These findings imply an important functional role for the ABC gene family. The identification of additional members of this family of genes or the demonstration that one or more of these genes are associated with specific human diseases has tremendous import to the better treatment and management of disease.
Analysis of amino acid sequence alignments of the ATP-binding domains has aided in separating the ABC genes into sub-families (Allikmets et al., Hum Mol Genet, 1996, 5, 1649-1655) according to the Human Genome Gene Organisation (HUGO) classification system. Seven ABC gene subfamilies named ABC A to G have been described in the human genome, e.g., ABCA (ABC1 subfamily), ABCB (MDR/TAP subfamily), ABCC(CFTR/MRP subfamily), ABCD (ALD subfamily), ABCE (OABP subfamily), ABCF (GCN20 subfamily), and ABCG (white subfamily). For the most part, these subfamilies contain genes that also display considerable conservation in the transmembrane domain sequences and have similar gene organization. However, ABC proteins transport various substrates, and some members of different subfamilies have been shown to share more similarity in substrate recognition than do proteins within the same subfamily. Several ABC transport proteins that have been identified in humans are associated with various diseases. Some multiple drug resistance phenotypes in tumor cells have been associated with the gene encoding the MDR (multi-drug resistance) protein, which also has an ABC transporter structure. Other ABC transporters have been associated with neuronal and tumor conditions (U.S. Pat. No. 5,858,719) or potentially involved in diseases caused by impairment of the homeostasis of metals (Biochim Biophys Acta. 1999; 1461 (2):18-404).
The human ABCC subfamily also currently has ten identified members (ABCC1 to 10), seven of which are from the multidrug resistance-like (MRP) subgroup, two from the sulfonylurea receptor (SUR) subgroup, and the CFTR gene. MRP-like proteins are organic anion transporters; e.g., they transport anionic drugs, exemplified by methotrexate (MTX), as well as neutral drugs conjugated to acidic ligands, such as glutathione (GSH), glucuronate, or sulfate, and play a role in resistance to nucleoside analogs (Cui et al., Mol Pharmacol, 1999, 55, 929-37; Kool et al., Proc Natl Acad Sci, 1999, 96, 6914-9; Schuetz et al., Nat Med, 1999, 5, 1048-51; Wijnholds et al., Proc Natl Acad Sci, 2000, 97, 7476-81). More specifically, ABCC1, ABCC2 and ABCC3 transport drugs conjugated to GSH, glucuronate, sulfate and other organic anions, such as MTX, whereas ABCC4 and ABCC5 proteins confer resistance to nucleotide analogs, including PMEA and purine base analogs.
Several genetic variations in some ABCC subfamily members have also been identified as associated with various human inherited diseases. For example, cystic fibrosis is caused by mutations in the ABCC7 gene or CFTR (cystic fibrosis transmembrane conductance regulator) gene (Riordan et al., Science, 1989, 245, 1066-73). Another member of the ABCC subfamily, the ABCC2 gene, has been associated with the Dubin-Johnson syndrome (Wada et al., Hum Mol Genet, 1998, 7, 203-7). Also, mutations in the coding sequence of another gene belonging to the ABCC subfamily, the ABCC6 gene, have been recently identified as responsible for the phenotype of pseudoxanthoma elasticum (Bergen et al., Nat. Genet., 2000, 25, 228-31; Le Saux et al., Nat Genet, 2000, 25, 223-7), which is a genetic disorder of the connective tissue. Likewise, a receptor of sulfonylureas, ABCC8 or SUR1, appears to be involved in familial persistent hyperinsulinemic hypoglycemia of infancy (Thomas et al., Science, 1995, 268, 426-9). The ABCC11 protein, as well as ABCC4 and ABCC5, is smaller than another well-known member of the subgroup, ABCC1 (MRP1), appearing to lack the extra N-terminal domain (Borst et al., J Natl Cancer Inst, 2000, 92, 1295-302), which is however not required for the transport function (Bakos et al., J. Biol. Chem, 1998, 273, 32167-75). Since structurally related ABC proteins often transport similar substrates across the membranes, it would be reasonable to suggest that the ABCC11 protein could share functional similarities with ABCC 4 and/or ABCC5 genes, e.g., the resistance to nucleotide analogs, such as PMEA, and purine base analogs (Schuetz et al., Nat Med, 1999 5, 1048-51; Wijnholds et al., Proc Natl Acad Sci, 2000, 97, 7476-81). Therefore, characterization of a new gene from the ABCC subfamily is likely to yield a biologically important transporter that may have a translocase activity and may play a major role in human pathologies.
The membrane-associated protein encoded by the ABCA5 is a member of the superfamily of ATP-binding cassette (ABC) transporters ABC1 subfamily (U.S. Patent Application Publication Nos. 2002/0123107 and 2003/0044895). It is clustered among 4 other ABC1 family members on 17q24 and it maps to chromosome 17q24.3 (Accession number: NM 172232) in Homo sapiens. Gene aliases include ABC13 and EST90625. This cluster of ABCA genes is evolutionarily distinct from that of other ABCA genes, as evidenced by phylogenetic analysis as well as analysis of intron-exon boundaries. The chromosome 17 ABCA genes have 38 exons, whereas the other ABCA genes have 50-52 exons. Therefore, it appears that all of the genes on chromosome 17 arose from an ancestral ABCA gene. This cluster is not represented in plant, nematode, or insect genomes, and there is a single ABCA5-related gene in fish. Thus, ABCA5 appears to be the ancestral gene for this cluster and seems to have arisen early in vertebrate evolution. ABCA5 is expressed as a 6.5-kb mRNA with the highest levels in the human trachea, prostate, testes, uterus and pancreas. Neither the substrate nor the function of the ABCA5 gene has been identified. Alternative splicing of this gene results in several transcript variants, but not all variants have been completely described. For example, transcriptional variant (NM—018672) represents the longer transcript variant. It contains a distinct 5′ UTR compared to transcript variant 2, but encodes the same protein.
Despite the foregoing evidence, a considerable controversy remains regarding the importance of PIN as a precursor of PCA. Thus, more work is required to confirm the relationship of PIN with the onset of PCA. More importantly, a specific marker for PIN is required in order to accurately detect PIN in patients. Furthermore, there exists a need in the art for an assay to diagnose PIN, prostate cancer and associated diseases or conditions. The present invention addresses and meets these needs.